Pyropheophorbide-based compounds have been developed as second generation photosensitizers (PS) that have low phototoxicity but high efficacy in photodynamic therapy (PDT) of various cancer forms, such as of the lung and aerodigestive tract. The goal of this project is to elucidate the cellular and molecular mechanisms by which these PS initiate signaling in epithelial tumor cells that leads to stress reactions and decision over cell death or survival. In the current funding period, diagnostic markers were identified that correlate in a dose-dependent fashion with the immediate cellular response to PDT. These markers include oxidative crosslinking of signal-transducing proteins, inactivation of cytokine and growth factor receptor functions, reduced cellular protein phosphorylation and activation of stress protein kinases. These changes are transient in PDT-surviving cells and, after a period of arrest, cells recover the capability to signal in response to inflammatory cytokines and resume proliferation. We have used these markers to identify newly designed derivatives of PS with enhanced uptake, altered subcellular distribution, increased singlet oxygen production and improved tumor control. We hypothesize that conjugation of PS with carbohydrates and metals alters PS uptake and subcellular accumulation resulting in enhanced cellular activity; that alterations in plasma membrane receptors and intracellular signaling play a major role in tumor cell survival following PDT; and that identification of regulatory mechanisms at multi-cellular level helps in devising combination therapies suppressing tumor, which survive the PDT reaction. Following specific aims are proposed to test these predictions: 1) To identify in mouse and human epithelial cells the mechanisms by which carbohydrate- and metal-conjugated PS achieve their cell type-specific action by determining uptake, cell organelle-preferred accumulation, and the relative contribution of ATP-binding cassette transporter-G2 to the steady state level of PS; 2) to define the peri- and post-PDT reactions within epithelial tumor cells that determine survival, recovery from stress, reestablishment of homeostasis, and resumption of proliferation; and 3) to develop a reconstituted three-dimensional co-culture system of epithelial, stromal or endothelial cells, which will permit the identification of the regulatory pathways that lead to the release of inflammatory mediators in response to PDT-treated tumor cells. The findings regarding PDT effect made in the tissue culture will be verified in tumors of preclinical animal models and in patients undergoing PDT treatment for skin and head/neck cancers as part of this program.